In recent years, the need for high-speed automated or semi-automated analysis and processing of biological cells and cell components has been recognized. Such analysis and processing may include a determination of morphologic characteristics of cells or differences in physical properties of cells, and is of special importance in the fields of cytochemistry, immunology, oncology, genetics, molecular biology and the like.
One method for analyzing and processing biological cells at high speed is flow and or scanning cytometry wherein the prepared cells are suspended in a carrier fluid and then are enclosed within an envelope, or sheath, stream and are passed one at a time through a sensing zone by hydrodynamic focusing. In the sensing zone, the cells are irradiated by a focused laser beam (with the cells being located outside of any laser cavity); and a light detector is used for the measurement of scattered, absorbed, or re-emitted fluorescent light. The effect that a cell has on the focused laser beam that it intercepts can be detected in a number of ways. In general, the cell has a refractive index which is different from that of the medium in which it is suspended. It will therefore scatter a portion of the laser light with which it is illuminated through a range of angles, and with varying intensifies, that depend upon the refractive index difference between the cell and the surrounding carrier fluid, the cell size and shape, and any internal variations in refractive index and structure in the cell, as well as the wavelength of the illuminating light. A cell may also absorb some of the incident light, with a portion of the absorbed light being re-emitted as fluorescence, typically at an emission wavelength that is longer than the wavelength of the absorbed light. Light detectors are arranged to measure different angular intervals of the scattered or fluorescent light.
Due to a low scattering efficiency of the small size of biological cells (typically less than 15 microns in diameter) and also a limited number of sites from which fluorescence may occur, the number of photons detected for each cell moving through the focused laser beam may be small, especially compared to the number of photons in the incident focused beam. Therefore, the limits of sensitivity of the prior art flow cytometry methods for cell analysis and processing depend critically on the photon flux (i.e. power) of the incident laser beam, and the magnitude of the perturbations in the scattered or fluorescent light produced by different variants of the biological cells to be analyzed (e.g. normal versus abnormal cells).
An advantage of the present invention is that biological cells may be analyzed and processed by locating the cells within an analysis region inside a laser cavity, with the cells acting in combination with a gain medium in the cavity to generate a laser beam having information about the cells impressed (i.e. encoded) thereupon.
Another advantage of the present invention is that information related to a size, shape, and type of cell, and internal characteristics (such as DNA, RNA, nucleohistones, mitochondria, golgi bodies, endoplasmic reticulum, lysozomes, and phagosomes) thereof may be impressed (i.e. encoded) upon a laser beam in the form of an emission spectrum, a transverse mode profile, an optical intensity, a nonlinear optical signal, a lasing threshold characteristic, or a combination thereof for providing information on a status of cell activation, cell proliferation, or cell life cycle.
A further advantage of the present invention is that information selective to a portion of a cell or to a particular constituent of the cell may be impressed upon a laser beam and recovered in an analysis means, for analyzing the cell and for subsequent processing thereof.
Still another advantage of the present invention is that a biological cell may be selectively tagged with a fluorescent stain or a non-fluorescent marker (e.g. a monoclinal antibody that may act to modify the cell structure and function), with the fluorescent stain forming at least a part of the gain medium of a laser cavity containing the cell; so that upon activation of the gain medium by optical pumping, lasing may be generated at predetermined locations on or within the cell wherein the florescent stain or marker is concentrated or localized.
Yet another advantage of the present invention is that a compact biological cell analyzer may be formed according to the present invention comprising on a substrate one or more analysis regions for containing biological cells wherein laser beams may be generated at the locations of particular cells, the laser beams providing information about the cells impressed (i.e. encoded) thereupon.
Another advantage of the present invention is that a compact biological cell analyzer and processor may be formed on a substrate having at least one inlet channel for introducing the cells substantially one at a time into one or more analysis regions wherein a laser beam is generated having information about the cells impressed thereupon, and a cell processing regions proximate to each analysis region wherein different variants of the cells may be separated after analysis into a plurality of reservoirs and/or outlet channels.
A further advantage of the present invention is that a carrier fluid (e.g. a buffered saline solution) surrounding the cell may contain one or more agents such as cell stimulants, drugs, or reagents that may act to modify cell properties; and these agents may be introduced into and/or flushed from the analysis region by flow channels for analyzing a response of the cell to these agents.
These and other advantages of the apparatus of the present invention will become evident to those skilled in the art.